An ideal HIV vaccine should provide protection against all HIV-1 variants. Thus, an important aspect of HIV vaccine development is the selection of immunogens, which has to take into consideration the diversity of the different HIV clades and the identification of the critical epitopes able to induce relevant immune responses, avoiding potential immunodominant decoy epitopes. Indeed, we reported a potent impact of Env on the induction of Gag cellular responses. To address these problems, we are exploring approaches to maximize immunological strength and breadth using mosaic and consensus molecules as well as focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant decoy epitopes. Using the latter, we have developed prototype vaccines targeting highly conserved regions within HIV Gag and Env. Immunogens were engineered encoding conserved elements (CE) of HIV-1 Gag selected on the basis of stringent conservation, functional importance, broad HLA-coverage, and association with viral control. In proof-of-concept studies in mice and macaques, we demonstrated that immunization with Gag CE DNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by vaccination with the full-length p55gag DNA. This vaccine induces robust cytotoxic T cell responses targeting subdominant epitopes. Importantly, we demonstrated that priming with CE DNA and boosting with p55gag DNA greatly augments the CE-specific responses and that inclusion of the p24CE+gag DNA in the boost maximizes both magnitude and breadth. Thus, we identified a novel and effective strategy to maximize responses against Gag and provide a novel strategy to shift the immunodominance hierarchy and to induce robust immune responses to subdominant epitopes, effectively targeting the Achilles' heel of the virus. We showed that this DNA vaccine regimen induces potent memory responses that can be rapidly recalled 2 years later by CE DNA booster vaccinations. The CE memory response can also be potently recalled by recombinant Modified Vaccinia Ankara (rMVA) booster vaccination, expression HIV Gag/Poland Env. These studies in macaques suggest application of this vaccine regimen in humans where DNA prime - rMVA boost has induced promising immunogenicity. We further developed an SIV homolog of the CE and demonstrated that priming with CE DNA followed by CE+gag DNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth. These data mirror our findings from the HIV p24CE vaccine and provide us with a tool to explore the functional applications of the Gag CE DNA vaccine in the macaque model. SIV Gag CE-specific T cells are able to reduce viral infection in an autologous in vitro virus inhibition assay, an important feature that has been shown to correlate with in vivo viral control in infected animals. The vaccine-induced CE-specific T cells were expanded upon SIV infection, indicating that the predicted CE epitopes incorporated in the DNA vaccine are processed and exposed by infected cells even in their natural context. Thus, our artificially generated immunogen containing linkers between the CE to optimize proteolytic processing and MHC association of the CE peptides, generates similar peptides as the virus infected cells. We found that CE responses contribute to control of viremia. Application of this CE DNA vaccine is expanded to our therapeutic vaccine trials in the macaque model. We have further expanded the CE concept to HIV Env. As found for HIV Gag CE, we reported that the identified CE in HIV Env are subdominant and that our novel DNA vaccine regimen by including a CE DNA prime alters the immune hierarchy and enable induction of robust cytotoxic T cell responses recognizing these epitopes. Together with the Gag CE DNA vaccine, this provides us with a vaccine to maximize magnitude and breadth of both Gag and Env. We are translating the novel HIV Gag CE DNA vaccine concept (CE DNA prime-CE+full length gag DNA boost) to the clinic in the HIV Vaccine Trial Network (HVTN)/DAIDS/NIAID-supported clinical trial (HVTN 119; opened October 4, 2017; NCT03181789) with the aim to test whether our p24CE DNA vaccine concept elicits superior breath and magnitude of Gag responses compared to the optimized immunogen comprising the complete p55Gag protein. This vaccine includes Profectus' GENEVAX IL-12 DNA as molecular adjuvant and in vivo electroporation as DNA delivery method, two vaccine components our research had shown to be of outmost importance to induce potent T cell responses in macaques. HVTN 119 will combine several of milestones (DNA expression vectors, adjuvants and delivery) we have achieved over many years in vaccine development. We are also translating the Gag CE DNA vaccine in a phase I/IIb trial (ACTG A5369; NCT03560258; planned opening August 17, 2018) supported by the AIDS Clinical Trial Group. In this trial, the CE DNA vaccine will be tested in HIV-infected persons under HAART with the aim to test safety and the immunogenicity of the HIV CE vaccine regimen in comparison to p55gag DNA vaccine, with breadth and magnitude of T cell responses as key endpoints. Although Antiretroviral Therapy (ART) has reduced HIV-related mortality and morbidity for infected individuals, a therapeutic regimen to eradicate HIV has not been achieved. Our goal is to develop and test immunotherapeutic methods that can lead to virus reservoir reduction or elimination. The induction of broad T cell responses targeting the subdominant highly conserved regions of the virus provides a unique possibility to explore our hypothesis that these highly cytotoxic T cell responses have bear the ammunition to target the Achilles's heel of HIV. Together, HVTN119 and A5369 trial will allow us to explore the translation of our data from mice and macaques to humans.